(596) Evaluating pre-existing immunity to CRISPR/Cas9 gene editing technologies

Background: Highly effective modulator therapies are able to restore the function of CFTR in ~90% of people with CF (PwCF), however the remaining PwCF harbor CFTR mutations that are not amenable to correction with the current therapies. While modulators significantly improve the quality of life and life expectancy of PwCF, they are not a long-lasting cure for CF. Advances in the development of delivery vectors provide a path for new attempts at gene therapy in CF. In addition, new gene editing technologies utilizing CRISPR/Cas9 have further advanced the possibilities to correct CFTR mutations ex vivo and in vivo. However, one potential challenge with CRISPR/Cas9 technologies is pre-existing host-immunity directed at Cas9 proteins, which are derived from bacterial species, including S. aureus that is a common CF pathogen. While recent studies have determined that non-CF healthy adults have pre-existing immunity to Cas9 proteins, this has not been investigated in PwCF. We hypothesize that PwCF will have increased correlates of immunity to Cas9 proteins when compared to non-CF controls due to chronic and recurrent infections.

Methods: To determine if PwCF have correlates of pre-existing immunity to CRISPR/Cas9, we isolated plasma from peripheral blood of PwCF and non-CF healthy donors. Serial dilutions of plasma were used in ELISAs for S. aureus or S. pyrogenes Cas9, tetanus toxin as a positive control, or human serum albumin (HSA) as a negative control, and the area under the curve was calculated for each sample.

Results: The ELISA-based protocol resulted in robust detection of reactivity to tetanus toxin, used as a positive control, and minimal background reactivity with HSA after calculation of AUC. Both recombinant Cas9 proteins from S. aureus or S. pyrogenes were determined to have significant increased detection over HSA. Our preliminary results demonstrate comparable AUC for all four analytes between non-CF and PwCF blood donors.

Conclusions: Our preliminary results suggest that pre-existing immunity to CRISPR/Cas9 gene editing technologies is present in PwCF as previously demonstrated for non-CF healthy donors. While our results have not uncovered significant differences in AUC between non-CF and PwCF, one potential limitation of the current study is the relatively small sample size. These results should be validated in larger populations of PwCF, and future studies will aim to further determine the prevalence and potency of T-cell mediated immunity to Cas9. Given the potential for utilizing CRISPR/Cas9 technologies for gene therapy in PwCF, performing similar measurements to determine the level of pre-existing immunity, and potential development of immunity following gene therapy delivery, should be incorporated into any human trials to help inform any results.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation grants LONG19F5-CI, LONG21R3 and MCCOY19R0. Recombinant Cas9 proteins were provided as a gift from IDT.

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